Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.     

Immunohistochemical localization of GABAA receptors in the scotopic pathway of the cat retina

The distribution of GABA_A receptors in the inner plexiform layer of cat retina was studied using monoclonal antibodies against the 2/3 subunits. A dense band of receptor labeling was found in the inner region of the inner plexiform layer where the rod bipolar axons terminate. Three forms of evidence indicate that the GABA_A receptor labeling is on the indoleamine-accumulating, GABAergic amacrine cell that is synaptically interconnected with the rod bipolar cell terminal. (1) Electron microscopy showed that the anti-GABA_A receptor antibody (62-3G1) labeled profiles that were postsynaptic to rod bipolar axons and made reciprocal synapses. (2) Indoleamine uptake (and the subsequent autofluorescence) combined with GABA_A receptor immunohistochemistry showed co-localization of the two markers in half of the receptor-positive amacrine cells. (3) Double labeling demonstrated that half of the receptor-positive somata also contained GABA. These results indicate that a GABAergic amacrine cell interconnected with the rod bipolar cell, most likely the so-called A17 amacrine cell, itself bears GABA_A receptors.     

The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) in species of Allium

Pulse labelling experiments with ³⁵SO₄ ^2- fed for 24h to intact plants (shooted onion sets)of Allium cepa (onion) showed that >70% of the label appeared in the S-alkenyl-L-cysteine sulphoxides within 18h, reached a maximum at 48h and thereafter decreased. The amount of label detected in the -glutamyl peptide fractions was below 20% of the total label at any time. It is concluded that in intact plants (at the growth stage used) the -glutamyl peptides are not the immediate precursors of the S-alkenyl-L-cysteine sulphoxides. The major S-alkenyl-L-cysteine sulphoxide in onion was found to be compartmentalized mainly within the endoplasmatic reticulum.Abbreviations AllCysSO (+)-S-2-propenyl-L-cysteine sulphoxide - MeCysSO (+)-S-methyl-L-cysteine sulphoxide - PrenCysSO trans-(+)-S-1-propenyl-L-cysteine sulphoxide - ProCysSO (+)-S-propyl-L-cysteine sulphoxide     

Distribution of catecholamines,indoleamines, and their precursors and metabolites in the scallop,Placopecten magellanicus (Bivalvia,Pectinidae)

Summary 1. Although monoamines are well-known to play important roles in molluscan physiology, we are far from fully understanding the synthetic and degradative pathways of these substances, particularly in commercially important bivalve species. In the present study endogenous catecholamines, indoleamines, and their possible precursors and metabolites were detected in the scallop,Placopecten magellanicus, by high-performance liquid chromatography coupled to electrochemical detection.2. Chromatographic analysis of CNS (cerebral, pedal, and parietovisceral combined), gill, gonad, kidney, mantle, liver, heart, fast adductor muscle, and foot disclosed the presence of the catecholamines 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, and epinephrine and their metabolites normetanephrine, metanephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid.3. Dopamine was detected most frequently and most consistently among all catecholamines. The concentrations of dopamine (1400 pg/mg wet weight) and its major metabolite 3,4-dihydroxyphenylacetic acid (300 pg/mg wet weight) were highest in the CNS. Following the CNS, dopamine was also abundant in other tissues such as heart, foot, and gill. The concentration of norepinephrine (1000 pg/mg wet weight) was highest in the CNS followed by the heart (700 pg/mg wet weight) and gill (600 pg/mg wet weight).4. The indoleamine, 5-hydroxytryptamine, was present in considerable amounts in all tissues, but its content was highest in the foot (2700 pg/mg wet weight) followed by the CNS (1150 pg/mg wet weight) and gonad (1000 pg/mg wet weight). The precursor 5-hydroxytryptophan was also abundant in the foot followed by the gonad, CNS, and heart.5. The oxidative metabolite 5-hydroxy-3-indole acetic acid was detected in the largest amount in CNS (200 pg/mg wet weight), whereasN-acetyl-5-hydroxytryptamine was detected in trace amounts in CNS, gonad and foot. This study also presents evidence for -glutamyl dopamine and -glutamyl 5-hydroxytryptamine as the possible alternate catabolic products of dopamine and 5-hydroxytryptamine, respectively, as previously described in gastropods.6. Thus, the detection of monoamines and their precursors and metabolites in scallop strongly suggests the presence of mammalian-type enzymic action of hydroxylation, oxidation, and methylation pathways leading to synthesis and degradation of detected compounds. Furthermore, this is the first study to disclose the evidence of nonconventional metabolic pathways for dopamine (-glutamyl dopamine dopamine dihydroxyphenylacetic acid homovanillic acid) and 5-hydroxytryptamine (-glutamyl 5-hydroxytryptamine 5-hydroxytryptamine 5-hydroxy-3-indoleacetic acid) inactivation in a bivalve species.     

Effects of nutrients and culture conditions on morphology in the seed culture ofCephalosporium acremonium ATCC 20339

The objective of this study was to investigate the effects of nutrients and culture conditions on morphology during the seed culture ofC. acremonium ATCC 20339. Morphological factors such as hyphal length, number of tips, number of arthrospores were observed to investigate the relationship between seed morphology and CPC production. During the time course of seed culture, hyphal length was shortened and the number of arthrospores increased rapidly. On the other hand, the number of tips decreased rapidly, and this was closely related to the hyphal length. Mixed nitrogen sources of 3% soybean meal and 1% cotton seed flour were determined as the proper organic nitrogen sources, in terms of the morphological factors in the seed culture. This fact was proven in batch culture for the production of Cephalosporin C. It was also found that a proper agitation speed enhanced the morphological differentiation ofC. acremonium ATCC 20339, thus improving the production of Cephalosporin C.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from typical and atypical strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.Abbreviations A-layer additional surface protein layer - A-protein surface protein - Ast Aeromonas salmonicida typical - Asa Aeromonas salmonicida atypical - A^- phenotypically A-protein-negative variant - O^- phenotypically O-polysaccharide-negative variant - O^wk phenotypically O-polysaccharide weak variant - BHI brain heart infusion - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TEM transmission electron microscopy     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.     

Release of acetylcholine and GABA,and activity of their synthesizing enzymes in the rat pontine reticular formation

The aim of this study was to obtain neurochemical information on the possible role of acetylcholine (ACh) and -aminobutyric acid (GABA) as neurotransmitters in the pontine reticular formation (PRF). We studied the uptake of labeled choline and GABA, as well as the release of this amino acid and of ACh, in PRF slices of the rat. In addition, choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase activities were assayed in PRF homogenates. The uptake of GABA was strictly Na⁺-dependent, whereas choline uptake was only partially Na⁺-dependent. The release of both ACh and GABA was stimulated by K⁺-depolarization, but only the former was Ca²⁺-dependent. Choline acetyltransferase activity in the PRF was 74% of that in the striatum, whereas acetylcholinesterase activity was considerably lower. Glutamate decarboxylase activity in the PRF was about half that observed in the striatum. These findings support the possibility that both ACh and GABA may act as neurotransmitters in the rat PRF.     

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence,effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rol, and -were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5 non-coding region of rol and rol was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.     

The wound response in the siphonous algaCaulerpa simpliciuscula C. Ag.: II. The effect of wounding on carbon flow

Summary Following on from a previous study on changes in cytology and fine structure during the wound response in the siphonous green algaCaulerpa simpliciuscula (Dreher, Grant, andWetherbee 1978), changes in the carbon metabolism during this wound response have been studied. There was a decrease in the rate of photosynthesis and an increase in the rate of respiration immediately on wounding, but rates of both photosynthesis and respiration returned to those of unwounded tissues within 6 hours. Wounding depressed the rate of starch synthesis and sucrose synthesis but increased the rate of synthesis of soluble 1,3 ß-glucan, lipid and sulphated polysaccharide. When the flow of carbon from these various compounds was studied by means of pulse chase experiments, it was found that only sucrose and sulphated polysaccharide showed different kinetics in control and wounded tissue. The changes which were observed are consistent with the direct involvement of sulphated polysaccharides in the formation of structures formed during the wound healing process.     

The dominant mutation Suppressor of black indicates that de novo pyrimidine biosynthesis is involved in the Drosophila tan pigmentation pathway

A deficiency in the production of -alanine causes the black (b) phenotype of Drosophila melanogaster. This phenotype is normalized by a semi-dominant mutant gene Su(b) shown previously to be located adjacent to or within the rudimentary (r) locus. The r gene codes for three enzyme activities involved in de novo pyrimidine biosynthesis. Pyrimidines are known to give rise to -alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to -alanine synthesis during the tanning process. In this report we show that flies carrying Su(b) can exhibit an additional phenotype, resistance to toxic pyrimidine analogs (5-fluorouracil, 6-azathymine and 6-azauracil). Our interpretation of this observation is that the pyrimidine pool is elevated in the mutant flies. However, enzyme assays indicate that r enzyme activities are not increased in Su(b) flies. Genetic mapping of the Su(b) gene now places the mutation within the r gene, possibly in the carbamyl phosphate synthetase (CPSase) domain. The kinetics of CPSase activity in crude extracts has been studied in the presence of uridine triphosphate (UTP). While CPSase from wild-type flies was strongly inhibited by the end-product, UTP, CPSase from Su(b) was inhibited to a lesser extent. We propose that diminished end-product inhibition of de novo pyrimidine biosynthesis in Su(b) flies increases available pyrimidine and consequently the -alanine pool. Normalization of the black phenotype results.     

Regulation of the appearance of glutamine synthetase in mustard (Sinapis alba L.) cotyledons by light,nitrate and ammonium

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS.The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor.Abbreviations c continuous - D darkness - FPLC fast protein liquid chromatography - GS glutamine synthetase (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) - FR far-red light (3.5 W·m^-2) - NF Norflurazon - R red light (6.8 W·m^-2, _R=0.8)) - RG9-light long-wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Production of specific-protein secretion granules by fat body cells of the blowfly,Calliphora erythrocephala

Summary During the first four days of the imaginai stage the fat cells of ovariectomized females of Calliphora develop a protein synthetic apparatus, and produce dense bodies (lysosomes) as do the fat cells of normal females, but apparently they cannot synthesize the protein secretion granules that characterize the productive phase of the fat cells of normal females and that we believe to represent vitellogenin. Injection of ovariectomized females with -ecdysone restored the ability of the fat cells to produce the secretion granules. It is suggested that the ovary gives off a factor which induces the production of the protein secretion granules by the fat cells, and that the factor from the ovary can be substituted by -ecdysone. This, we believe, is the first ultrastructural evidence for an effect of the ovary and of -ecdysone on the synthesis of specific protein.We are grateful to the Carlsberg Foundation and to the Danish Science Research Council for generous grants, and to the latter for placing an electron microscope at our disposal. It is a pleasure to thank Dr. Gareth Griffiths for valuable advice as to the preservation of the fat body tissue. We also thank Mrs. Lotte Bakhoj and Mrs. Elsebeth Lund for skilful technical assistance1896–1976     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody     

Mechanism involved in the response of granulated vesicles in the mouse pinealocyte to acute cold exposure

Summary Rudimentary cilia have been observed in muscle cells lining the tube feet of Ophioderma brevispinum (Ophiuroidea) and in muscle cells of the body wall and parapodial glands of Owenia fusiformis (Polychaeta). A diplosomal basal body is associated with each cilium. Striated rootlets are absent. This is the first report on rudimentary cilia in muscle cells of an echinoderm and an annelid.Supported by NSF grant #GB-42211 to R.M. Rieger. Costs of some photographic supplies were defrayed by a grant from Sigma Xi to S.L. Gardiner